Summary Approximately 65,000 patients in the United States alone develop empyema (EMP) or complicated parapneumonic pleural effusions each year. The incidence of EMP is increasing worldwide. EMP is associated with serious morbidity, a mortality of about 20%, and annual patient care costs of roughly $500 million. Intrapleural fibrinolytic therapy (IPFT) has been used for over sixty years to expedite pleural drainage and prevent lung restriction, but its efficacy and safety profile, especially in adults, remains uncertain. Bleeding occurs in up to 15% of patients. Current IPFT protocols use empirically dosed, off-label interventions and reflect rudimentary knowledge about the regulation of IPFT in EMP and its pathogenesis. These gaps have slowed the development of more reliable and safer IPFT for patients with EMP or other forms of loculated pleural injury and form the scientific premise for our project. Our preliminary data validates active plasminogen activator inhibitor 1 (PAI-1) as a biomarker and therapeutic target for IPFT. We provide proof of concept and show that three mechanistically different forms of PAI-1 targeted IPFT effectively reverse tetracycline-induced pleural organization in rabbits. These interventions allow for up to an 8-fold reduction of the dose of fibrinolysin in IPFT, thereby mitigating bleeding risk. We designed a companion diagnostic test; the Fibrinolytic Potential Assay (FPA), to monitor outcomes of PAI-1-targeted or other forms of IPFT. Our hypothesis is that PAI-1 targeted IPFT can effectively clear intrapleural organization in EMP. We also posit that FPA can predict outcomes of PAI-1-targeted IPFT and ultimately be used to select the subjects most likely to benefit. To test this hypothesis, we developed and characterized a new S. pneumoniae rabbit EMP model which simulates key features of human EMP from an acute (96h) to an organized chronic (7-21 days) phase with multiloculation-like organization and significant pleural thickening. Our objective is to test the efficacy and safety of PAI-1 targeted IPFT in the S. pneumoniae EMP model and determine if the FPA predicts pleural injury outcomes. This hypotheses will be tested in four Specific Aims, which are to: 1. Improve the efficacy of IPFT in rabbit EMP using PAI-1 targeted delivery; 2. Determine the molecular mechanisms governing intrapleural fibrinolysis in EMP and the effects of high levels of extracellular DNA on PAI-1 targeted IPFT; 3. Select the single most effective form of PAI-1 targeted IPFT in EMP, develop and validate the companion FPA test using EMP fluids from the model and patients; and 4. Optimize the structure and co-formulation of a novel IPFT for EMP and evaluate its safety and efficacy. A range of state-of-the-art biochemical, physiologic, tissue analysis, and imaging techniques will be used to accomplish the work. Our unique multidisciplinary team includes leading experts in the fields of fibrinolysis, pleural injury, as well as drug formulation and development. This project addresses current unmet needs: the identification and preclinical vetting of new, potentially more effective and safer IPFT candidates and FPA diagnostics for EMP that are clinically tractable and that could ultimately improve clinical outcomes.